1. Field of the Invention
This invention relates to fluorescence spectrophotometers and, in particular, to spectrophotometric instruments capable of generating and analyzing both emission and absorption spectra.
2. Description of the Prior Art
Conventional fluorescence spectrophotometers of early vintage comprised, in their essential constituency, a source of exciting radiant energy, usually in the ultraviolet range of the spectrum; a monochromator for converting the radiant energy to a range of wavelengths utilized to irradiate the specimen undergoing analysis and cause fluorescent emissions; and a detection system, including a second monochromator, for measurement of the intensity of fluorescent radiation emitted by the specimen. Normally, the monochromators were of the scanning type capable of generating a range of wavelengths of a continuous sequence so that the specimen could be scanned with a band of exciting wavelengths and the response to each such wavelength detected and compared and/or recorded.
The basic instrument described above suffered a major shortcoming; its results were susceptible to fluctuations of the radiation source (commonly an xenon lamp) and variations in the spectral characteristics of the monochromators. This shortcoming was overcome, at least in part, by the adoption of an optical system utilizing a reference beam of radiation and a comparison or ratio recording system. The excitation energy emanating from the first (or "excitation") monochromator was passed through a sample cell holding the specimen and thence to a detector, which generated an electrical signal processed through a signal amplifier and one channel of a ratio recorder. Simultaneously, the fluorescent emission of the sample was directed to another (the "emission") monochromator, then to a detector and subsequently, in the form of an electrical signal, through a signal amplifier to the second channel of the recorder. In this manner, fluctuations in the radiation source and variations in the spectral characteristics of the monochromators appeared in both reference and specimen channels and were cancelled from the reading.
The efficacy of the use of a reference beam to obtain a true excitation spectrum was, however, incomplete because, as can be demonstrated mathematically, its functioning depends on equality in the intensity of radiation at the respective wavelengths involved, impinging on the specimen and on the detector cell (e.g., a thermocouple, bolometer, rhodamine B fluorescent or the like). In practice, the irradiation reaching the reference detector passes through and has its intensity diminished by absorption in the specimen cell.
In order to cope with this problem, spectrum correction systems have been devised utilizing light quantum meters (or quantum counters) employing high concentration solutions of rhodamine B. A corrected excitation and emission system for fluorescence analysis is described, for example, by Poro, Anacreon, Flandreau, and Fagerson in "Corrected Fluorescence Spectra. . ." appearing in the Journal of the Association of Official Analytical Chemists, Vol. 56, No. 3, 1973 at pages 607-610.
In spectrophotometric analysis, it is highly desirable to utilize both emission and absorption spectra for comparison purposes. The validity of the comparison, of course, depends in large measure on the comparability of the instruments on which the analysis is carried out. Disparities, of course, are elminated or minimized if the emission and absorption spectral analyses are carried out on the same instrument and that capabilities of that instrument are equal for both types of analysis.
Unfortunately, the fluorescence spectrophotometers evolved to date, capable of obtaining a true excitation and emission spectra, are capable of only single beam operation when used for absorption spectrum analysis. This detracts from the desired accuracy of comparison with the true excitation spectrum analysis.
One solution, of course, would be to utilize separate instruments, one for the emission analysis, and the other for absorption. Aside from the added expense of a self-recording double-beam spectrometer for the absorption spectrum analysis, a precise comparison of results is difficult because, regardless of the similarity in quality and design of the instruments, they are nevertheless distinct entities which makes identical analytical parameters, e.g., slit conditions, practically impossible to achieve.
With the foregoing state of the art in view, it is the basic general object of the present invention to provide a novel instrument for spectroscopic analysis of both emission and absorption spectrum.
A more particular object is the provision of an instrument as characterized in the proceeding object in which the absorption spectrum analysis as well as true or corrected emission spectrum analysis is performed with a double beam system and the analyses are in all respects directly comparable in accuracy and precision.
Another object is the provision of a method for performing spectroscopic analysis of both fluorescene emission and absorption spectra on a single instrument with only minor modification of the optical system employed.